1. What is the turnaround time?
The entire RPPA process takes approximately 8-10 weeks to complete. This timeframe accounts for sample accumulation and sample processing time. Once we accumulate 1,000 samples from customers to accommodate a full set, the RPPA processing time is approximately 4 weeks. Service is provided on a "first come, first served" basis; note that “first come” refers to samples received and requires a fully approved iLab request and arranging for sample drop off or shipping with the RPPA Core. We encourage you to submit your online forms and samples as soon as possible.
2. How should I prepare my samples? Do you have special protocols?
Yes, we have protein extraction protocols posted on the Resources page. You can also purchase lysis buffer and 4× SDS sample buffer from the Core through iLab. The charge is $1.50/ml (+ applicable overhead). Please contact core personnel to schedule a pickup or shipment of the buffers. For shipping, we require you to provide a valid FedEx number for shipping charges.
3. After completion, what information and data can I expect to receive from the Core?
Your dataset will be arranged in an Excel file. It includes raw data in log2 values and normalized data in linear and log2 values. You will be able to generate bar graphs or other formatted graphs using normalized linear values. We also provide heatmaps generated from median-centered log2 values. These heatmaps represent visualization figures for your reference.
Please consult your bioinformatics group for further detailed analysis. (MD Anderson users click consult Bioinformatics Shared Resources)
4. How do you normalize the data set?
We perform 2 sets of normalization procedures: (1) normalization for protein loading for all samples within a set (Level 3 data) and (2) debatching across sets by replicates-based normalization (RBN, Level 4 data). Unlike Western blots where only one protein is used (e.g., β-Actin or GAPDH), we use the entire panel of antibodies to normalize for protein loading. Normalization for Level 3 data involves bidirectionally median centering a table consisting of rows of all 1056 samples and columns of all reported antibodies. (See example)
RBN involves normalizing by taking identical (technically replicated) control samples in the given set with an invariant control sample set and applying the adjustment (differences in means × inverse of standard deviation ratio) to each corresponding data point to produce Level 4 data. Level 4 data enables comparison of results across different RPPA Core sets. (Click on RBN for more details.)
The median of each antibody median-centered sample in Level 3 data generation is referred to as “Total Protein Content” (TPC) and reflects the sample’s average signal across the entire antibody panel. Low values reflect low average signal of the sample while high values reflect high average signal. Any sample with a TPC <-3 is considered too low for reliable quantification results. We recommend carefully reviewing or censoring these results.
5. How do we quantify protein expression and modification?
We use a “supercurve fitting” approach developed by the Department of Bioinformatics and Computational Biology at MD Anderson Cancer Center to quantify protein expression and modification. Briefly, a "standard curve" is constructed from 5808 spots on each slide (one slide probed for one antibody). These spots include 5 serial dilutions from each sample plus 528 QC spots of standard lysates at different concentrations. Relative levels of protein expression and modification for each sample are determined by interpolation of each dilution curve to the "standard curve" (supercurve) of the slide (antibody). (Click on RPPA SPACE for more information)
6. If I have several RPPA data sets that were performed at different times within different RPPA assays, can I combine all my data together for further analysis?
The replicates-based normalization (RBN) process enables comparison of samples in sets containing Level 4 data. We have generated Level 4 data since Set 183.
Please note RBN utilizes technical replicates of our control samples so while these control samples are debatched, other samples might have conditions that debatching of controls cannot overcome. In any case, you should design constant internal controls that will be included within each experiment to be used for data merging in future RPPA assays.
7. I have samples ready but they were prepared in the buffer for my western blot. Can I submit these samples for RPPA?
We encourage you to use the lysis buffer with our formulation. However, we accept samples prepared by the approach for Western blot. Our experience demonstrates that samples prepared in RIPA buffer will work for RPPA. However, samples prepared from Urea buffer will make diffused spots that interfere with quantification.
8. What is the optimal protein concentration of the samples for RPPA? If my sample concentration is below 1 ug/ul, can you run this sample for RPPA?
The optimal protein concentration is 1 µg/µl after adding 4× SDS buffer. We accept samples with a minimum concentration of 0.50 µg/µl from cell lines and 0.75 µg/µl from tissue. If you have a few samples with low protein concentration in your sample set, we suggest that you not to adjust protein concentration against the lowest one. Otherwise, you will lose most of the signals.
9. How much volume of each sample do you need for RPPA?
We require a minimum volume of 80 µl per sample to accommodate our entire panel of about 500 antibodies.
10. Do I need to prepare replicate samples for RPPA?
There are two kinds of replicates: biological replicates and technical replicates. Please design replicates according to your study. Please note we charge each replicate as a separate sample. We perform multiple technical replicates using our standard lysates across the slides on different positions. These replicates serve as quality controls for each antibody in RPPA. We report the results of antibodies that pass our quality controls, but it is your discretion if and how many replicates you choose to submit. If you submit replicates, we encourage using biological replicates as technical replicates have no real benefit.
11. Do I need to select antibodies for RPPA?
No, we have a standard panel of antibodies that we use for each set. Please refer to the Resources page. You do not need to select antibodies from our Expanded Panel; we will perform the entire panel of antibodies on this list and release the results from the antibodies that passed our quality control.
12. If the antibody against the protein of my interest does not exist in your list, can I provide you with an antibody?
Yes, you can provide your own antibodies of interest. You need to provide 100 µl of the antibody along with full length Western blot results (if applicable) when you submit your samples. Please contact the Core to determine whether your antibody has already been validated and if it has not, it will need to be validated prior to use. We will provide you with a validation report detailing its validation status after completion. The validation status will be judged on the following criteria:
A. good correlation between RPPA and mass spectrometry or RPPA and RNA expression and
B. further confirmation by Western blot.
Criteria for Western blot includes:
- a single band on Western blot with the correct molecular size,
- acceptable dynamic range on the specific protein,
- good correlation between Western blot and RPPA (r > 0.7), and
- acceptable signal to noise ratio on RPPA. We will also probe your samples with your validated antibody. We will only use antibodies that have a validation status of "Valid" or "Use with Caution" according to our standard validation criteria.
Please note that since we do not have experience with your antibody and have not optimized its use for our RPPA application, we cannot guarantee the result.
Charges apply for these services. Please refer to the Services page for details.
13. If I would like you to validate my antibody, do you provide such a service?
Yes. Please see Question #12.
14. I have a set of tissue samples to be submitted for RPPA analysis. How much tissue do you need from each sample? Do you have protocols to extract proteins from tissue?
We require at least 15 mg of frozen tissue (approximately the size of a large grain of rice) per sample with at least 60% cellularity for processing the entire panel of antibodies. We prefer that your tissue samples be prepared in our lysis buffer.
15. Do you provide service to extract protein from tissue or cell pellets for RPPA?
Yes, we provide service for protein extraction from fresh frozen tissue and dry frozen cell pellets. We currently do not accept FFPE samples. We require at least 15 mg of tissue (about the size of a large grain of rice) with at least 60% cellularity or cell pellets containing 1-5 million cells, depending on the cell size (about a 5 µl pellet).
16. I have a set of clinical samples. Can I submit them for RPPA analysis?
Yes. You must delete patient health information (PHI) such as the medical record number, patient name, etc., before you submit. We will not accept samples with PHI.
17. I am in the process of submitting a grant application and/or manuscript that will involve RPPA analysis. Do you have any information I can refer to in my grant application?
Yes. Please refer to the Acknowledgement page.
18. I am a research investigator outside of MD Anderson Cancer Center. How should I pay for the RPPA service?
You need to complete the online submission process through iLab Solutions and include a valid purchase order number or complete the credit card authorization form. Full payment is due no later than 30 days after receipt of the invoice.
19. Do I need to validate my result from RPPA?
Yes. We consider RPPA a screening method for discovery. You need to validate and confirm your results by other technical approaches.